Method of purifying proteins

ABSTRACT

The present invention relates generally to a method of purifying proteins. More specifically, the present inventions relates to a method of purifying haptoglobin and hemopexin from the same starting material, and uses thereof.

CROSS REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication No. 61/709,342, filed Oct. 3, 2012, which is herebyincorporated by reference.

TECHNICAL FIELD

The present invention relates generally to a method of purifyingproteins. More specifically, the present inventions relates to a methodof purifying haptoglobin and hemopexin from the same starting material,and uses thereof.

BACKGROUND

Haemolysis is characterized by the destruction of red blood cells and isa hall-mark of anaemic disorders associated with red blood cellabnormalities, such as enzyme defects, haemoglobinopathies, hereditaryspherocytosis, paroxysmal nocturnal haemoglobinuria and spur cellanaemia, as well as extrinsic factors such as splenomegaly, autoimmunedisorders (e.g., Hemolytic disease of the newborn), genetic disorders(e.g., Sickle-cell disease or G6PD deficiency), microangiopathichaemolysis, Gram-positive bacterial infection (e.g., Streptococcus,Enterococcus and Staphylococcus), parasite infection (e.g., Plasmodium),toxins and trauma (e.g., burns). Haemolysis is also a common disorder ofblood transfusions, particularly massive blood transfusions and inpatients using an extracorporeal cardio-pulmonary support.

The adverse effects seen in patients with conditions associated withhaemolysis are largely attributed to the release of iron andiron-containing compounds, such as haemoglobin (Hb) and heme, from redblood cells. Under physiological conditions, released haemoglobin isbound by soluble proteins such as haptoglobin and transported tomacrophages and hepatocytes. However, where the incidence of haemolysisis accelerated and becomes pathological in nature, the bufferingcapacity of haptoglobin is overwhelmed. As a result, haemoglobin isquickly oxidised to ferri-haemoglobin, which in turn releases free heme(comprising protoporphyrin IX and iron). Whilst heme plays a criticalrole in several biological processes (e.g., as part of essentialproteins such as haemoglobin and myoglobin), free heme is highly toxic.Free heme is a source of redox-active iron, which produces highly toxicreactive oxygen species (ROS) that damages lipid membranes, proteins andnucleic acids. Heme toxicity is further exacerbated by its ability tointercalate into lipid membranes, where is causes oxidation of membranecomponents and promotes cell lysis and death.

The evolutionary pressure of continuous low-level extracellular Hb/hemeexposure has led to compensatory mechanisms that control the adverseeffects of free Hb/heme under physiological steady-state conditions andduring mild haemolysis. These systems include the release of a group ofplasma proteins that bind Hb or heme, including the Hb scavengerhaptoglobin (Hp) and the heme scavenger proteins hemopexin (Hx) andα1-microglobulin. However, whilst endogenous Hp and Hx control theadverse effects of free Hb/heme under physiological steady-stateconditions, they have little effect in maintaining steady-state Hb/hemelevels under pathophysiogical conditions, such as those associated withhaemolysis.

The present invention provides a method of purifying Hp and Hx from thesame starting material. The purified proteins can be used incompositions for treating conditions associated with haemolysis andaberrant Hb/heme levels.

SUMMARY OF THE INVENTION

In an aspect of the present invention, there is provided a method ofpurifying haptoglobin and hemopexin from a solution containing bothproteins, the method comprising:

-   -   (i) providing a solution containing both haptoglobin and        hemopexin;    -   (ii) precipitating the haptoglobin from the solution by adding        ammonium sulphate to the solution;    -   (iii) separating the precipitated haptoglobin from the solution        containing hemopexin; and    -   (iv) separately purifying the haptoglobin and/or hemopexin in        one or more steps.

In another aspect of the present invention, there is provided acomposition comprising the haptoglobin recovered by the methodsdisclosed herein.

In another aspect of the present invention, there is provided acomposition comprising the hemopexin recovered by the methods disclosedherein.

In another aspect of the present invention, there is provided acomposition comprising the transferrin recovered by the methodsdisclosed herein.

In another aspect of the present invention, there is provided acomposition comprising the haptoglobin recovered by the methodsdisclosed herein and the hemopexin recovered by the methods disclosedherein.

In another aspect of the present invention, there is provided acomposition comprising a haptoglobin content of at least 80% of totalprotein. In another aspect of the present invention, there is provided acomposition comprising a hemopexin content of at least 80% of totalprotein. In another aspect of the present invention, there is provided acomposition comprising a combined hemopexin and haptoglobin content ofat least 80% of total protein.

In another aspect of the present invention, there is provided aformulation comprising the composition of the present invention, asdisclosed herein, and a pharmaceutically acceptable carrier.

In another aspect of the present invention, there is provided a methodof treating a condition associated with haemolysis, the methodcomprising administering to a subject in need thereof the composition orthe formulation of the present invention, as disclosed herein.

In another aspect of the present invention, there is provided use of thecompositions or formulations of the present invention, as disclosedherein, in the manufacture of a medicament for treating a conditionassociated with haemolysis.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a flow diagram of a Cohn Fractionation Process.

FIG. 2 shows the recovery of transferrin (TRF), Albumin (Alb), hemopexin(HPX) and haptoglobin (HAP) in the remaining filtrate followingprecipitation in the presence of 2.0M, 2.5M, 3.0M, 3.5M and 4.0Mammonium sulfate.

FIG. 3 shows a desirability plot of the design of experiment (DOE),showing the desirable conditions that would precipitate the haptoglobinfrom the plasma fraction, while keeping the hemopexin in solution. A DOEis a set of controlled experimentation that was used to evaluate theimpact of pH and ammonium sulfate concentration on the ability toseparate hemopexin from haptoglobin. A factorial mathematical design wasused to design and analyze the data. The desirability plot depicted inFIG. 3 uses this mathematical design to determine the most desirableconditions that would result in the best separation and recovery of bothhemopexin and haptoglobin.

FIG. 4 shows SDS-PAGE electrophoresis of hemopexin recovered followingthe various steps in the purification process disclosed herein. SDS-PAGEanalysis was performed using pre-cast 10% Tris-Glycine gels. All sampleswere diluted to a concentration of 0.1 mg/mL in Tris-Glycine SDS samplebuffer and 20 uL of each sample was loaded into the sample well of thegel. Run time and Voltage were set to the gel manufacturer'srecommendations. Each gel was stained with an easy to use type ofCoomassie Brilliant Blue stain solution purchased direct from amanufacturer.

FIG. 5 shows an SDS-PAGE electrophoresis (using the method describedabove in FIG. 4) of haptoglobin intermediates recovered following thevarious steps in the purification process disclosed herein.

FIG. 6 shows a Western Blot of the Capto Q Eluate on a 10% Tris-GlycineNon-Reduced SDS-PAGE electrophoresis gel (using the method described inFIG. 4). The separated proteins are then transferred to a nitrocellulosemembrane and the membrane is blocked to prevent any non-specific bindingof antibody. The nitrocellulose membrane is then incubated with asolution containing antibodies to Human Haptoglobin. A secondaryantibody linked to horseradish peroxidase is then incubated with thenitrocellulose membrane. The nitrocellulose is then developed with asolution containing peroxide thereby only visualizing the protein bandsthat specifically contain Human Haptoglobin. All lanes contain Capto QImpRes Eluate at different concentrations. The Western blot indicatesthat most of the bands present in the Capto Q Eluate are haptoglobin.

FIG. 7 shows SDS-PAGE electrophoresis of transferrin recovered fromion-exchange chromatography (Capto DEAE) following the various steps inthe purification process disclosed herein. Peak one is heavily loaded,but appears to be pure transferrin. This indicates that it is possibleto purify transferrin from the Octyl Sepharose Wash fraction, which alsomeans that it is possible to purify hemopexin, haptoglobin, andtransferrin from the same starting material.

FIG. 8 is a flow diagram of a hemopexin purification process inaccordance with an embodiment disclosed herein.

FIG. 9 is a flow diagram of a haptoglobin purification process inaccordance with an embodiment disclosed herein.

FIG. 10 is a flow diagram of a combinedhaptoglobin/hemopexin/transferrin purification process in accordancewith an embodiment disclosed herein.

DETAILED DESCRIPTION

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

The reference in this specification to any prior publication (orinformation derived from it), or to any matter which is known, is not,and should not be taken as an acknowledgment or admission or any form ofsuggestion that that prior publication (or information derived from it)or known matter forms part of the common general knowledge in the fieldof endeavour to which this specification relates.

It must be noted that, as used in the subject specification, thesingular forms “a”, “an” and “the” include plural aspects unless thecontext clearly dictates otherwise. Thus, for example, reference to “aresin” includes a single resin, as well as two or more resins; referenceto “the composition” includes a single composition, as well as two ormore compositions; and so forth.

In the absence of any indication to the contrary, reference made to a“%” content throughout this specification is to be taken as meaning %w/w (weight/weight). For example, a solution comprising a haptoglobincontent of at least 80% of total protein is taken to mean a compositioncomprising a haptoglobin content of at least 80% w/w of total protein.

The present invention is predicated, at least in part, on the findingthat haptoglobin and hemopexin can be purified from the same startingmaterial. Thus, in an aspect of the present invention, there is provideda method of purifying haptoglobin and hemopexin from a solutioncontaining both proteins, the method comprising:

-   -   (i) providing a solution containing both haptoglobin and        hemopexin;    -   (ii) precipitating the haptoglobin from the solution by adding        ammonium sulphate to the solution;    -   (iii) separating the precipitated haptoglobin from the solution        containing hemopexin; and    -   (iv) separately purifying the haptoglobin and/or hemopexin in        one or more steps.

Haptoglobin (Hp) is a tetrachain (α₂β₂) glycoprotein synthesized by theadult liver and secreted into the plasma. The propeptide form of Hp isproteolytically cleaved into an α-chain and a β-chain. Two α-subunitsand two β-subunits of Hp protein are then joined by inter-chaindisulfide bonds to form the mature peptide, which can be either an(αβ)-dimer or an (αβ)-multimer. Hemopexin (Hx) is a 60-kD plasmaβ-1B-glycoprotein comprising a single 439 amino acid long peptide chain,which forms two domains joined by an interdomain linker. It has thehighest known affinity for heme (Kd<1 pM) of any characterizedheme-binding protein and binds heme in an equimolar ratio between thetwo domains of Hx in a pocket formed by the interdomain linker.

The inventors have found that an ammonium sulfate concentration in therange of about 2.0M to about 2.5M, preferable in the range of about 2.2Mto about 2.5M, more preferably about 2.4M, is optimal for separatingboth proteins from the same starting material. Thus, in an embodiment,the method comprises precipitating the haptoglobin from the solution byadding about 2.0M to about 2.5M ammonium sulphate to the solution. Inanother embodiment, the method comprises precipitating the haptoglobinfrom the solution by adding about 2.2M to about 2.5M ammonium sulphateto the solution. In yet another embodiment, the method comprisesprecipitating the haptoglobin from the solution by adding about 2.4Mammonium sulphate to the solution.

The inventors have also shown that a pH maintained in the range of lessthan or equal to about 8, preferably about 6 to about 8, more preferablyat about 7.0, is optimal for separating both proteins from the samestaring material. Thus, in an embodiment, the method comprisesprecipitating the haptoglobin from the solution at a pH of less than orequal to 8. In another embodiment, the method comprises precipitatingthe haptoglobin from the solution at a pH within the range of about 6 toabout 8. In yet another embodiment, the method comprises precipitatingthe haptoglobin from the solution at a pH of about 7.

In the methods disclosed herein, the majority of haptoglobin from thestarting material will be found within the ammonium sulfate precipitateand the majority of the hemopexin from the starting material will befound in the remaining solution (also referred to as the suspension).However, persons skilled in the art will understand that the precipitatemay comprise some hemopexin (e.g., trace amounts of Hx) and that theremaining solution (or suspension) may comprise some haptoglobin (e.g.,trace amounts of Hp). Where trace amounts of Hp and Hx are present inthe suspension and precipitate, respectively, it may be desirable toremove these by separately purifying the haptoglobin and/or hemopexin inone or more steps in accordance with the methods disclosed herein.However, persons skilled in the art would understand that trace amountsof Hp and Hx that may be present in the suspension and precipitate,respectively, may be acceptable, for example, where both proteins willend up in the same composition.

Any solution comprising both haptoglobin and hemopexin can be used asthe staring material in the method of the present invention, disclosedherein. Suitable starting material would be known to persons skilled inthe art, examples of which include plasma fractions such as varioussupernatants and precipitates derived from ethanol fractionationprocesses. Examples of such ethanol fractionation processes include Cohnfractionation and Kistler-Nitschmann fractionation. Examples of suitableplasma fractions include those derived from a Cohn fraction I, II, III,II+III, I+II+III, IV and V (See FIG. 1) or a Kistler-Nitschmann fractionsuch as a Precipitate A or B. In an embodiment, the solution is a humanplasma fraction. In another embodiment, the solution is a Cohn FractionIV. In yet another embodiment, the solution is a Cohn Fraction IV₄. In aparticularly preferred embodiment the solution is derived from aFraction IV₄ Precipitate.

It will be understood that, where the starting material is provided as aprecipitate (e.g., Fraction IV₄ Precipitate), it will be necessary toinitially resolublise the precipitate to provide a suitable startingsolution for the methods of the present invention.

The methods of the present invention are suitable for thecommercial/industrial scale purification of hemopexin, haptoglobin and,optionally, transferrin. For example, when using plasma fractions as astarting material, employing the method of the present invention on acommercial/industrial scale may involve the use of a plasma fractionderived from at least about 500 kg of plasma. More preferably, theplasma fraction will be derived from at least about 5,000 kg, 7,500 kg,10,000 kg and/or 15,000 kg of plasma per batch.

The skilled person will understand that plasma for fractionation is theliquid part of blood remaining after separation of the cellular elementsfrom blood collected in a receptacle containing an anticoagulant, orseparated by any other suitable means known to persons skilled in theart, such as by continuous filtration or centrifugation ofanticoagulated blood in an apheresis procedure.

In an embodiment, the precipitated haptoglobin and the solutioncontaining hemopexin are recovered and stored separately beforeseparately purifying the haptoglobin and/or hemopexin in one or moresteps, in accordance with the present invention. In another embodiment,the precipitated haptoglobin and the solution containing hemopexin arerecovered and subjected immediately to further purification steps inaccordance with the methods of the present invention; that is,separately purifying the haptoglobin and/or hemopexin in one or moresteps.

Thus, in an embodiment, the method further comprises:

-   -   (i) dissolving the precipitated haptoglobin in a buffer to        obtain a haptoglobin solution;    -   (ii) passing the haptoglobin solution through a strong anion        exchange chromatographic resin under conditions such that the        haptoglobin binds to the resin;    -   (iii) eluting the haptoglobin from the resin; and    -   (iv) recovering the eluted haptoglobin.

Purification of proteins by chromatography can be performed using eitheraxial flow columns, such as those available from GE Healthcare, PallCorporation and Bio-Rad, or using radial flow columns, such as thoseavailable from Proxcys. Chromatography can also be conducted usingexpanded bed technologies known to persons skilled in the art.

Most chromatographic processes employ a solid support, also referred tointerchangeably herein as a resin or matrix. Suitable solid supportswould be familiar to persons skilled in the art and the choice willdepend on the type of product to be purified. Examples of suitable solidsupports include inorganic carriers, such as glass and silica gel,organic, synthetic or naturally occurring carriers, such as agarose,cellulose, dextran, polyamide, polyacrylamides, vinyl copolymers ofbifunctional acrylates, and various hydroxylated monomers, and the like.Commercially available carriers are sold under the names of Sephadex™,Sepharose™, Hypercel™, Capto™, Fractogel™, MacroPrep™, Unosphere™,GigaCap™, Trisacryl™, Ultrogel™, Dynospheres™, Macrosorb™ and XAD™resins.

The chromatography steps will generally be carried out undernon-denaturing conditions and at convenient temperatures in the range ofabout −10° C. to +30° C., more usually at about ambient temperatures.The chromatographic steps may be performed batch-wise or continuously,as convenient. Any convenient method of separation may be employed, suchas column, centrifugation, filtration, decanting, or the like.

Buffers that are suitable for dissolving the haptoglobin precipitatewould be familiar to persons skilled in the art and may depend on theconditions required for performing the chromatographic purificationstep. Examples of suitable buffers are sodium acetate and Tris with a pHrange of 5.5 to 9.0. Particular embodiments utilize a pH of 7.5 to 9.0however, in a preferred embodiment the buffer is about 50 mM Tris at apH of about pH 8.4 to about pH 8.6.

In embodiments the lipid content of the extracted precipitate comprisinghaptoglobin is reduced by exposure to a lipid removal agent underconditions that allow the lipid to bind to the lipid removal agent.Examples of such agents include fumed silica such as Aerosil. In apreferred embodiment the lipid removal agent is Aerosil. The lipidremoval agent such as Aerosil can be added to the extracted precipitatecomprising haptoglobin at about 0.5 g to about 4 g per liter of plasmaequivalent. In particular embodiments, Aerosil is added at 1 to 2 g perliter plasma equivalent. In a preferred embodiment Aerosil is added at1.6 g per liter of plasma equivalent. It was determined that lipidremoval is most effective within a specific pH range. A pH range of 5.5to 8.5 was found to be effective in conjunction with Aerosil. Thepreferred embodiment utilizes a pH range of 8.4 to 8.6.

The lipid removal agent can be removed using methods such as filtrationand or centrifugation. In particular embodiments the lipid removal agentis removed by depth filtration. An example of a depth filter for use inthis application is a Cuno 70CA filter or one of similar or smallerparticle size retention capabilities.

Persons skilled in the art will understand that any strong anionexchange chromatographic resin can be used to separately purifyhaptoglobin from the haptoglobin solution, as long as the haptoglobin iscapable of binding to the chromatographic resin while allowing someimpurities in the solution to pass though the resin. Persons skilled inthe art would also determine that due to the ionic strength of theextraction buffer and subsequent pH adjustment of the load solution;dilution, diafiltration, or other methods of buffer exchange/ionicstrength reduction would be required to allow haptoglobin to bind to theresin. Suitable resins would be known to persons skilled in the art.Examples of suitable anion exchange resins are ones comprising afunctional quaternary amine group (Q) and/or a diethylaminopropyl group(ANX). In an embodiment, the strong anion exchange chromatographic resincomprises a functional quaternary amine group (e.g., Capto Q ImpRes™).

Buffers that are suitable for eluting the haptoglobin from the resinwill also be known to persons skilled in the art. An example includessodium acetate. Particular embodiments utilize 50 mM sodium acetate at apH of 5.0 to 6.0. In a preferred embodiment the buffer is about 50 mMsodium acetate at a pH of about pH 5.3 to about pH 5.7.

In further embodiments, the haptoglobin is eluted from the anionexchange resin with an elution buffer comprising from about 100 mM toabout 200 mM NaCl. This equates to an elution buffer having aconductivity range of about 10 mS/cm (100 mM NaCl) to about 18 mS/cm(200 mM NaCl). In particular embodiments the haptoglobin is eluted inthe presence of about 150 to 170 mM NaCl. In a preferred embodiment thehaptoglobin is eluted in the presence of about 160 mM NaCl.

In an embodiment, the eluted haptoglobin is recovered and storedseparately for future use. In another embodiment, the eluted haptoglobinis further purified, for example, by concentrating and diafiltering theeluted haptoglobin through an ultrafiltration membrane and/or sterilefiltering the concentrated and/or diafiltering haptoglobin, as required.

In an embodiment, the method further comprises:

-   -   (i) passing the solution containing hemopexin through a        hydrophobic interaction chromatographic resin under conditions        that allow the hemopexin to bind to the resin;    -   (ii) collecting the flow-through fraction from step (i);    -   (iii) optionally washing the resin following step (ii) and        collecting the flow-through wash fraction; and    -   (iv) eluting the hemopexin from the resin following step (ii)        and/or following step (iii); and    -   (v) recovering the eluted hemopexin.

Hydrophobic Interaction Chromatography (HIC) is a chromatographictechnique frequently used for the separation of proteins on the basis ofa hydrophobic interaction between the stationary phase and the proteinto be separated. The level of hydrophobicity of the target protein willoften dictate the type of HIC resin to be used. During HIC, a highamount of salt is typically added to the solution to reduce thesolubility of the target protein and thus increase the interaction ofthe target protein with the HIC resin functionalized with a suitablehydrophobic groups (e.g., phenyl, butyl and octadecyl groups). Suitablehydrophobic interaction chromatographic resins would be familiar topersons skilled in the art. An example includes an octyl sepharosechromatographic resin. The conditions that allow the hemopexin to bindto the resin will be known to persons skilled in the art and will bedictated, for example, by the type of resin used and the hydrophobicityof the target protein (i.e., hemopexin).

Once the solution containing hemopexin is passed through the hydrophobicinteraction chromatographic (HIC) resin, the flow through fraction canbe collected and stored for future use, as disclosed herein.

The bound hemopexin can be eluted from the resin by means known topersons skilled in the art. Prior to eluting the hemopexin from theresin, the resin can optionally be washed with a suitable wash solutionor buffer under conditions that retain the hemopexin bound to the resin.Suitable wash solutions and conditions will be known to persons skilledin the art. The flow through wash fraction can also be collected andstored for future use, as necessary.

The eluted hemopexin that is recovered from the resin can be stored forfuture use. The eluted hemopexin may also be subjected to furtherpurification to remove any impurities in the eluate. Thus, in anembodiment, the method further comprises:

-   -   (i) passing the eluted hemopexin through a metal ion affinity        chromatographic resin under conditions that allow the hemopexin        to bind to the resin; and    -   (ii) eluting the hemopexin from the resin; and    -   (iii) recovering the eluted hemopexin.

Immobilized metal ion affinity chromatography (IMAC) is based on thecovalent attachment of amino acids (e.g., histidine) to metals, allowingproteins with an affinity for metal ions to be retained in a columncontaining immobilized metal ions, such as zinc, cobalt, nickel orcopper. Suitable metal ion affinity chromatographic resins would beknown to persons skilled in the art. In an embodiment, the metal ionaffinity chromatographic resin is Ni-Sepharose.

Once the hemopexin is bound to the metal ion affinity chromatographic(IMAC) resin, the resin may be washed to remove any residual impuritiesunder conditions that retain the hemopexin bound to the resin. The boundhemopexin can be eluted from the resin by means known to persons skilledin the art. The eluted hemopexin can be further purified, for example,by concentrating and diafiltering the hemopexin through anultrafiltration membrane and/or sterile filtering the concentratedand/or diafiltering hemopexin, as required.

The inventors have also found that any transferrin that may be presentin the starting material remains in solution (i.e., in the solutioncomprising hemopexin) following the precipitation of haptoglobin in thepresence of ammonium sulfate. Thus, the methods of the presentinvention, disclosed herein, can also be used to purify transferrin fromthe same starting material. Conditions are therefore provided that areoptimal for purifying all three proteins (Hp, Hx and transferrin) fromthe same starting material. Thus, in an embodiment, the solutioncontaining both haptoglobin and hemopexin (e.g., the starting material)will further comprise transferrin.

In an embodiment, the method further comprises:

-   -   (a) passing the solution containing hemopexin through a        hydrophobic interaction chromatographic resin under conditions        that allow the hemopexin to bind to the resin;    -   (b) collecting the flow-through fraction from step (a);    -   (c) optionally washing the resin following step (b) and        collecting the flow-through wash fraction;    -   (d) passing the flow-through fraction from step (b) and/or the        flow-through wash fraction from step (c) through a weak anion        exchange chromatographic resin under conditions such that        transferrin binds to the resin; and    -   (e) recovering the transferrin from the resin.

Suitable weak anion exchange chromatographic resins will be known topersons skilled in the art. Examples include resins comprising atertiary or secondary amine functional group, such as DEAE(diethylaminoethyl).

Once the transferrin is recovered from the weak anion exchangechromatographic resin, it can be further purified, for example, byconcentrating and diafiltering the transferrin through anultrafiltration membrane and/or sterile filtering the concentratedand/or diafiltering transferrin, as required.

Where a solution comprising haptoglobin and/or hemopexin and/ortransferrin is to be used for clinical or veterinary applications (e.g.,for administration to a subject with a condition associated withhaemolysis), persons skilled in the art will understand that it may bedesirable to reduce the level of active virus content (virus titre) andother potential infectious agents (for example prions) in the solution.This may be particularly desirable where the feedstock comprisinghaptoglobin and/or hemopexin and/or transferrin (i.e., the startingmaterial) is derived from blood plasma. Methods of reducing the virustitre in a solution will be known to persons skilled in the art.Examples include pasteurization (for example, incubating the solution at60° C. for 10 hours in the presence of high concentrations ofstabilisers such as glycine (e.g. 2.75M) and sucrose (e.g. 50%) and/orother selected excipients or salts), dry heat treatment, virusfiltration (passing the solution through a nano-filter; e.g., 20 nmcutoff) and/or subjecting the solution to treatment with a suitableorganic solvent and detergent for a period of time and under conditionsto inactivate virus in the solution. Solvent detergent has been used forover 20 years to inactivate enveloped viruses particularly inplasma-derived products. Thus it may be carried out using variousreagents and methods known in the art (see, for example, U.S. Pat. No.4,540,573 and U.S. Pat. No. 4,764,369 which are hereby incorporated byreference). Suitable solvents include tri-n-butyl phosphate (TnBP) andether, preferably TnBP (typically at about 0.3%). Suitable detergentsinclude polysorbate (Tween) 80, polysorbate (Tween) 20 and Triton X-100(typically at about 0.3%). The selection of treatment conditionsincluding solvent and detergent concentrations depend in part on thecharacteristics of the feedstock with less pure feedstocks generallyrequiring higher concentrations of reagents and more extreme reactionconditions. A preferred detergent is polysorbate 80 and a particularlypreferred combination is polysorbate 80 and TnBP. The feedstock may bestirred with solvent and detergent reagents at a temperature and for atime sufficient to inactivate any enveloped viruses that may be present.For example, the solvent detergent treatment may be carried out forabout 4 hours at 25° C. The solvent detergent chemicals are subsequentlyremoved by for example adsorption on chromatographic media such as C-18hydrophobic resins or eluting them in the drop-through fraction of ionexchange resins under conditions which adsorb the protein of interest.

The virus inactivation step can be performed at any suitable stage ofthe methods disclosed herein. In an embodiment, the feedstock comprisinghaptoglobin and/or hemopexin and/or transferrin is subject to a viralinactivation step prior to step (ii) from the first described aspect. Inanother embodiment, the solution comprising haptoglobin and/or hemopexinand/or transferrin that is recovered from the ammonium sulphateprecipitation step (i.e., from steps (ii) and/or (iiii)) is subject to aviral inactivation step. In an embodiment disclosed herein, the viralinactivation step comprises pasteurisation or treatment with an organicsolvent and detergent. In another embodiment disclosed herein, the virusinactivation step comprises virus filtration. Where virus filtration isused, the inventors have found that the addition of a free amino acid(e.g., arginine) prior to the filtration step can significantly improvethe flux rate and recovery of haptoglobin and/or hemopexin and/ortransferrin through the filter. An example of such method is describedin U.S. Pat. No. 7,919,592.

In an embodiment disclosed herein, the feedstock or solution comprisinghaptoglobin and/or hemopexin and/or transferrin is subject to a viralinactivation step before it is passed through a chromatographic resin.The advantage of employing a virus inactivation step such as solventdetergent treatment prior to passing the treated solution or feedstockthrough a chromatographic resin such as an anion exchange resin is thatit allows for the removal of the organic solvent and detergent from thetreated solution by utilizing conditions that promote binding of thehaptoglobin and/or hemopexin and/or transferrin to the resin and removalof the organic solvent and detergent with the flow-through(drop-through) fraction.

Pasteurization can generate protein aggregates and polymers. Therefore,it may be desirable in some instances to reduce the level ofaggregates/polymers in a pasteurized solution. This can be achieved byany means knows to persons skilled in the art, although conveniently canbe achieved by further chromatographic purification. In an embodimentdisclosed herein, the pasteurized solution or feedstock is passedthrough an anion exchange chromatographic resin in positive mode withrespect to the haptoglobin and/or hemopexin and/or transferrin such thatany aggregates or polymers are removed with the flow-through(drop-through) fraction.

In another aspect of the present invention, there is provided acomposition comprising the haptoglobin recovered by the methodsdisclosed herein. In an embodiment, the composition comprises ahaptoglobin content of at least 80% of total protein. In anotherembodiment, the composition comprises a haptoglobin content of at least90% of total protein. In another embodiment, the composition comprises ahaptoglobin content of at least 95%. In yet another embodiment, thecomposition comprises a haptoglobin content of at least 98%.

In another aspect of the present invention, there is provided acomposition comprising the hemopexin recovered by the methods disclosedherein. In an embodiment, the composition comprises a hemopexin contentof at least 80% of total protein. In another embodiment, the compositioncomprises a hemopexin content of at least 90% of total protein. Inanother embodiment, the composition comprises a hemopexin content of atleast 95%. In another embodiment, the composition comprises a hemopexincontent of at least 97%. In yet another embodiment, the compositioncomprises a hemopexin content of at least 98%.

In another aspect of the present invention, there is provided acomposition comprising the transferrin recovered by the methodsdisclosed herein. In an embodiment, the composition comprises atransferrin content of at least 80% of total protein. In anotherembodiment, the composition comprises a transferrin content of at least90% of total protein. In another embodiment, the composition comprises atransferrin content of at least 95%. In yet another embodiment, thecomposition comprises a transferrin content of at least 98%.

In another aspect of the present invention, there is provided acomposition comprising the haptoglobin recovered by the methodsdisclosed herein and the hemopexin recovered by the methods disclosedherein. In an embodiment, the composition comprises a combinedhaptoglobin and hemopexin content of at least 80% of total protein. Inanother embodiment, the composition comprises a combined haptoglobin andhemopexin content of at least 90% of total protein. In anotherembodiment, the composition comprises a combined haptoglobin andhemopexin content of at least 95% of total protein. In yet anotherembodiment, the composition comprises a combined haptoglobin andhemopexin content of at least 98% of total protein.

In an embodiment, the composition further comprises the transferrinrecovered by the methods disclosed herein. In an embodiment, thecomposition comprises a combined haptoglobin, hemopexin and transferrincontent of at least 80% of total protein. In another embodiment, thecomposition comprises a combined haptoglobin, hemopexin and transferrincontent of at least 90% of total protein. In another embodiment, thecomposition comprises a combined haptoglobin, hemopexin and transferringcontent of at least 95% of total protein. In yet another embodiment, thecomposition comprises a combined haptoglobin, hemopexin and transferrincontent of at least 98% of total protein.

In another aspect of the present invention, there is provided acomposition comprising a haptoglobin content of at least 80%, 90%, 95%,or 98% of total protein. In another aspect of the present invention,there is provided a composition comprising a hemopexin content of atleast 80%, 90%, 95%, or 98% of total protein. In another aspect of thepresent invention, there is provided a composition comprising a combinedhemopexin and haptoglobin content of at least 80, 90%, 95%, or 98% oftotal protein. In yet another aspect of the present invention, there isprovided a composition comprising a combined hemopexin, haptoglobin andtransferrin content of at least 80, 90%, 95%, or 98% of total protein.

The compositions comprising haptoglobin, hemopexin and/or transferrinrecovered by the methods of the present invention disclosed herein willbe substantially free of other components with which they are normallyassociated (e.g., other plasma-derived proteins). Thus, in anembodiment, the composition comprising haptoglobin, hemopexin and/ortransferrin will comprise less than 20% of total protein, preferablyless than 10% of total protein, and more preferably less than 5% oftotal protein of other components with which they are normallyassociated (i.e., impurities). The skilled person will understand thatthe level of impurities present in the compositions of the presentinvention may depend on the intended use of the compositions. Forexample, where the compositions are to be administered to a humansubject in need thereof (i.e., for clinical use), it would be desirablethat the composition comprises less than 5% impurities (of totalprotein). Conversely, where the proteins are to be used in vitro, it maybe acceptable if the composition comprises more than 5% of impurities(of total protein).

In another aspect of the present invention, there is provided aformulation comprising the composition of the present invention, asdisclosed herein, and a pharmaceutically acceptable carrier.

Suitable pharmaceutically acceptable carriers, diluents and/orexcipients are known to those skilled in the art. Examples includesolvents, dispersion media, antifungal and antibacterial agents,surfactants, isotonic and absorption agents and the like.

The pharmaceutical formulation may also be formulated by the addition of(or a combination of) suitable stabilisers, for example, an amino acid,a carbohydrate, a salt, and a detergent. In particular embodiments, thestabiliser comprises a mixture of a sugar alcohol and an amino acid. Thestabilizer may comprise a mixture of a sugar (e.g. sucrose ortrehalose), a sugar alcohol (e.g. mannitol or sorbitol), and an aminoacid (e.g. proline, glycine and arginine). In a preferred embodiment,the formulation comprises an amino acid such as arginine. In otherembodiments, the formulation comprises divalent metal ions in aconcentration up to 100 mM and a complexing agent as described in U.S.Pat. No. 7,045,601. In embodiments where the pH is preferably about 6.5to 7.5 and the osmolality is at least 240 mosmol/kg.

The pharmaceutical formulation may also be sterilised by filtrationprior to dispensing and long term storage. Preferably, the formulationwill retain substantially its original stability characteristics for atleast 2, 4, 6, 8, 10, 12, 18, 24, 36 or more months. For example,formulations stored at 2-8° C. or 25° C. can typically retainsubstantially the same molecular size distribution as measured byHPLC-SEC when stored for 6 months or longer. Particular embodiments ofthe pharmaceutical formulation can be stable and suitable for commercialpharmaceutical use for at least 6 months, 12 months, 18 months, 24months, 36 months or even longer when stored at 2-8° C. and/or roomtemperature.

The compositions described herein may be formulated into any of manypossible dosage forms such as injectable formulations. The formulationsand their subsequent administration (dosing) are within the skill ofthose in the art. Dosing is dependent on the responsiveness of thesubject to treatment, but will invariably last for as long as thedesirable effect (e.g., a reduction in the level of free Hb/heme) isdesired. Persons of ordinary skill can easily determine optimum dosages,dosing methodologies and repetition rates.

In an embodiment disclosed herein, the pharmaceutical formulation of thepresent invention is a solution that has a volume of at least 5 mL andcomprises at least 5 mg/mL haptoglobin and/or hemopexin and/ortransferrin. In another embodiment, the pharmaceutical formulation has avolume of at least 5 mL and comprises at least 20 mg/mL haptoglobinand/or hemopexin and/or transferrin. In particular embodiments, thepharmaceutical formulation has a volume of at least 5 mL and compriseshaptoglobin and/or hemopexin and/or transferrin at a concentration ofabout 20 mg/mL, 25 mg/mL, 30 mg/mL, 35 mg/mL, 40 mg/mL, 45 mg/mL, 50mg/mL, 55 mg/mL, 60 mg/mL, 65 mg/mL, 70 mg/mL, 75 mg/mL, 80 mg/mL, 90mg/mL, 100 mg/mL, 150 mg/mL or 200 mg/mL. In another aspect, there isprovided a vessel containing at least 5 mL of a stable pharmaceuticallyacceptable haptoglobin and/or hemopexin and/or transferrin solution,wherein the concentration of haptoglobin and/or hemopexin and/ortransferrin is at least 20 mg/mL.

In another aspect of the present invention, there is provided a methodof treating a condition associated with haemolysis, the methodcomprising administering to a subject in need thereof the composition orthe formulation of the present invention, as disclosed herein.

The term “subject”, as used herein, refers to an animal which includes aprimate (a lower or higher primate). A higher primate includes human.Whilst the present invention has particular application to targetingconditions in humans, it would be understood by those skilled in the artthat non-human animals may also benefit from the compositions andmethods disclosed herein. Thus, it will be appreciated by the skilledaddressee that the present invention has both human and veterinaryapplications. For convenience, an “animal” includes livestock andcompanion animals such as cattle, horses, sheep, pigs, camelids, goats,donkeys, dogs and cats. With respect to horses, these include horsesused in the racing industry as well as those used recreationally or inthe livestock industry.

The compositions or formulations of the present invention may beadministered to the subject a number of ways. Examples of suitableroutes of administration include intravenous, subcutaneous,intra-arterial or by infusion. In an embodiment, the molecules areadministered intravenously.

Where necessary, the methods of the present invention may furthercomprise administering a second therapeutic agent. The secondtherapeutic compound may be co-administered to the subject sequentially(before or after administration of the compositions or formulationsdisclosed herein) or concurrently. In an embodiment, the secondtherapeutic agent is an iron chelating agent (e.g., deferrioxamine ordeferiprone).

In another aspect of the present invention, there is provided use of thecompositions or formulations of the present invention, as disclosedherein, in the manufacture of a medicament for treating a conditionassociated with haemolysis. Such compositions or formulations arepreferably suitable for use in human patients.

Conditions associated with haemolysis and which are at risk ofhaemoglobin/heme-mediated toxicity, are known in the art. In anembodiment, the condition is selected from an acute haemolytic conditionand/or a chronic haemolytic condition. In an embodiment, the conditionis selected from the group consisting of haemolytic anaemia,transfusion-induced haemolysis, haemolytic uraemic syndrome, anautoimmune disease, malaria infection, trauma, blood transfusion, openheart surgery using cardiopulmonary bypass and burns, including in thetreatment of hemoglobinemia or hemoglobinuria accompanied with hemolysisafter burn. In an embodiment, the condition is selected from the groupconsisting of sickle cell anaemia, hereditary spherocytosis, hereditaryelliptocytosis, thalassemia, congenital dyserythropoietic anemia andParoxysmal nocturnal hemoglobinuria, systemic lupus erythematosus andchronic lymphocytic leukemia.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications which fall within thespirit and scope. The invention also includes all of the steps,features, compositions and compounds referred to or indicated in thisspecification, individually or collectively, and any and allcombinations of any two or more of said steps or features.

Certain embodiments of the invention will now be described withreference to the following examples which are intended for the purposeof illustration only and are not intended to limit the scope of thegenerality hereinbefore described.

EXAMPLES Example 1

Starting Material: Cohn Fraction IV4 Precipitate was used as startingmaterial for the purification of haptoglobin, hemopexin, and transferrin(see FIG. 1).

Precipitate Extraction: Extraction of the precipitate was performed bythe introduction of 20 grams of Extraction Buffer per gram of FractionIV4 Precipitate (20× Extraction Ratio). The buffer and the precipitatewere mixed for a minimum of 1 hour. Extraction Buffer consisted of 50 mMTris adjusted to a pH of 7.0 with Concentrated HCl. The ExtractionBuffer was prepared at a temperature of 20-25° C. The precipitateextraction was also performed at a temperature of 20-25° C. The pHduring the extraction was maintained between 7.0 to 8.0 (preferably 7.0)for the duration of the 1 hour extraction time.

Ammonium Sulfate Precipitation: Solid ammonium sulfate was added to theFraction IV4 extract in order to achieve a final concentration of 2.0 to2.5M (preferably 2.4M). Under agitation, the ammonium sulfate was slowlyadded to the extract and allowed to continually mix for a minimum of 1hour. The ammonium sulfate precipitation was performed at a temperatureof 20-25° C.

An ammonium sulfate concentration of 2.5M was utilized to precipitatelipids, and clarify the Fraction IV4 Extract, while keeping hemopexinand transferrin soluble. Coincidentally, this ammonium sulfateconcentration resulted in the precipitation of a significant portion ofthe haptoglobin present in the Fraction IV4 Extract (see FIG. 2). Theprecipitation of haptoglobin at 2.2M to 2.5M ammonium sulfate, whilehemopexin remained soluble, allows for the co-purification of hemopexinand haptoglobin from the same starting fraction.

To further narrow the acceptable ammonium sulfate concentration range, adesign of experiment (DOE) was performed which looked at the impact ofpH and ammonium sulfate concentration. FIG. 3 shows the desirabilityplot of the DOE. This plot gives the most desirable conditions thatwould precipitate the most haptoglobin while keeping the most hemopexinsoluble. The desirability plot shows that a pH maintained between 7-8(preferably 7.0) and an ammonium sulfate concentration between 2.2M and2.5M (preferable 2.4M) is optimal for separating both proteins from thesame staring material. Moreover, transferrin remains soluble atconcentrations greater than 2.5M, hence, these conditions were optimalfor the purification of all three proteins from the same startingmaterial.

Filtration: To prepare the ammonium sulfate treated extract forfiltration, 10 grams of C1000 filter aid was added per litre of plasmaequivalent utilized for the batch. The C1000 filter aid was allowed tomix for a minimum of 15 minutes prior to the execution of filtration. Aplate and frame filter press was utilized for the filtration to allowfor collection of the haptoglobin-enriched precipitate. The plate andframe filter press was assembled with a sheet of type 175 filter paperin front of a 3M (Cuno) 70CA depth filtration filter sheet. For every 3L of plasma equivalent input into the batch, 0.193 mL of precipitatecollection area was required (3 L Plasma/4″ Ertel 4S Filter Frame). Theammonium sulfate treated extract was then pumped into the filter pressthrough the use of a double diagram pressurized air actuated pump.

After the completion of the C1000 mix time, the treated extract waspumped into the filter press and the filtrate was collected after asingle pass through the filter press. The filter press was thenpost-washed with 1 to 2 press volumes of 2.4M ammonium sulfate, 50 mMTris, adjusted to pH 7.0. The filtration process was performed at atemperature of 20-25° C. The resulting filtrate contains hemopexin andcan be stored at 2-8° C. until it is carried forward for furtherpurification. The resulting precipitate contains haptoglobin and can bestored at less than or equal to −20° C. until it is carried forward forfurther purification.

Purification of Hemopexin (Hx) from the Filtrate Fraction:

The Hx process scheme is highlighted in FIG. 8.

(a) Octyl Sepharose Chromatography (HIC): The filtrate obtained from theextraction and ammonium sulfate treatment of Fraction IV4 precipitatewas further clarified using a 0.22 μm filter. The filtrate was thenloaded onto an Octyl Sepharose column (GE Lifesciences) that had beenequilibrated with three column volumes of 2.5M ammonium sulfate, 50 mMTris, at pH 7.4. Eight to 12 column volumes (Target=10) of filtrate wereloaded onto the Octyl Sepharose column Impurities and any unboundprotein were washed off of the column with 3 column volumes of 0.9M to1.2M (Target 1.13M) ammonium sulfate, 50 mM Tris, at pH 7.4 (Wash). Thewash fraction contained transferrin, which can be saved for furtherpurification. The hemopexin containing fraction (Eluate) was then elutedoff of the column with three column volumes of water (WFI). The Eluatewas then stored at 2-8° C. until used for further purification. TheOctyl Sepharose (HIC) purification was performed at a temperature of20-25° C.

(b) Ni-Sepharose Chromatography (IMAC): 20 mM sodium phosphate, 500 mMsodium chloride, and 30 mM imidazole were added to the Octyl SepharoseEluate. Once added, the pH of the Octyl Sepharose Eluate was adjusted to7.4. Two to 3 column volumes of the Octyl Sepharose Eluate were thenloaded onto a Ni-Sepharose (GE Lifesciences) column that had beenequilibrated with buffer containing 20 mM sodium phosphate, 500 mMsodium chloride and optionally 30 mM imidazole, adjusted to pH 7.4. Theaddition of imidazole to the Octyl Sepharose Eluate (load) reduces theaffinity of the Ni-Sepharose to albumin and other impurities, whilemaintaining its binding affinity to hemopexin. Therefore, during theload step, the impurities flowed through the column, while the hemopexinbound to the resin. After the completion of the load, the column waswashed with 2 column volumes of 20 mM sodium phosphate, 500 mM sodiumchloride and 30 mM imidazole, pH 7.4 to remove any unbound impurities.The hemopexin was then eluted from the column using 20 mM sodiumphosphate, 500 mM sodium chloride and 100 mM imidazole, at pH 7.4. Thehemopexin present in the Ni-Sepharose eluate was estimated to be greaterthan 95% by SDS-PAGE (see FIG. 4). The Ni-Sepharose chromatography(IMAC) process was performed at 20-25° C. and the resulting eluate wasstored at less then or equal to −20° C. until use.

(c) Concentration/Diafiltration: The Ni-Sepharose Eluate wasconcentrated to a desired concentration (1-20% w/v), then diafilteredwith 10 volumes of phosphate buffered saline per volume of concentrate.The concentration and diafiltration were performed using a 30 kDultrafiltration membrane. Once the diafiltration was completed and theconcentrate was at the desired concentration, the hemopexin wasoptionally formulated with a sugar and or amino acid, sterile filteredand stored at less than or equal to −20° C. The purified hemopexin canbe in pre-clinical animal and cellular studies in this form.

The final yield of hemopexin recovered from the process was estimated tobe approximately 0.151 g/L plasma.

The concentrated hemopexin preparation (approximately 2.3% w/v) wascharacterised by immune-nephelometry. Plasma proteins such as IgG, IgA,IgM, alpha-1-antitrypsin, transferrin, alpha-1-acid glycoprotein,pre-albumin, ceruloplasmin, apolipoprotein A-I, apolipoprotein B andantithrombin III were below detectable levels. Only trace amounts weredetected for other plasma proteins, such as haptoglobulin (0.165 mg/mL)and albumin (0.038 mg/mL). These results indicate that Hx accounted forat least 99% of the total protein in the preparation.

A further batch of hemopexin was processed according to the methodsdescribed above. The batch was concentrated to 35 mg/mL protein.Analysis of the batch indicated a hemopexin purity of about 98% with theimpurities including 1.6% haptoglobin, 0.3% transferrin, 0.2% albumin,and 0.1% alpha-2 macroglobulin.

Purification of Haptoglobin (Hp) from the 2.5M Ammonium SulfatePrecipitate

The Hp process scheme is highlighted in FIG. 9.

(a) Precipitate Extraction and Filtration: Haptoglobin was extractedfrom the precipitate by the introduction of 20 grams of extractionbuffer/gram of precipitate (20× ratio). The extraction buffer consistedof 50 mM Sodium Acetate, adjusted to pH 5.5. The buffer and theprecipitate were mixed for a minimum of 1 hour. After 15 minutes, the pHwas adjusted to within the range of 5.4 to 5.6. The low pH extractionbuffer was utilized to reduce lipid extraction while the low pH wasutilized during the subsequent chromatography step. To remove remainingfilter aid and undissolved protein and lipids, the extract was thenpassed through a Cuno 70CA filter or equivalent depth filter. Thefiltrate was then clarified by use of a 0.2 μm filter. The resultingfiltrate was ready for the subsequent chromatography step. Theextraction buffer preparation, the extraction process, and thefiltration process were all performed at 20-25° C.

In another embodiment the Haptoglobin was extracted from the precipitateby the introduction of 20 grams of extraction buffer/gram of precipitate(20× ratio). The extraction buffer consisted of 50 mM Tris, adjusted topH 8.5. The buffer and the precipitate were mixed for a minimum of 1hour. After 15 minutes, the pH was adjusted to within the range of 8.4to 8.6. To reduce the lipid content and aid in clarification of theextracted precipitate a lipid adsorption agent, Aerosil (fumed silica),was added at 1.6 grams per liter of plasma equivalent. The Aerosiltreated extract was then allowed to mix for a minimum of 1 hour. Toremove remaining filter aid and any undissolved protein and lipids, theextract was passed through a Cuno 70CA filter or other similar depthfilter. The filtrate was then clarified by use of a 0.2 μm filter. Theresulting filtrate was ready for the subsequent chromatography step. Theextraction buffer preparation, the extraction process, and thefiltration process were all performed at 20-25° C.

(b) Capto Q ImpRes Chromatography Step: Sodium acetate was added to thefiltrate obtained from the above step, to a final concentration of 50mM, and the pH of the filtrate is adjusted to pH 5.5. The pH adjustedfiltrate was then diluted 1:4 to 1:5 with water (WFI) and loaded onto aGE Healthcare Capto Q ImpRes chromatography column that was equilibratedwith 50 mM sodium acetate, pH 5.5. The load requires sodium acetate as alow pH buffer and dilution with water is required in order to reduce theconductivity of the load so that haptoglobin can bind to the columnAfter completion of the load, the column was washed with 2 columnvolumes of 50 mM sodium acetate, pH 5.5 to remove any unboundcontaminate proteins. The haptoglobin was then eluted with 4 columnvolumes of 50 mM sodium acetate, 100-200 mM NaCl (preferably 162 mM), pH5.5. A wide NaCl concentration range of the elution buffer is requiredas the elution conditions are partially dependant on the load volumeutilized. The eluate can be stored at 2-8° C., short term, and at lessthan or equal to −20° C., long term, until concentration/diafiltration.The haptoglobin content of the Capto Q ImpRes eluate was estimated to begreater than 95% by SDS-PAGE (see FIGS. 5 and 6). The chromatographybuffers and chromatography steps were all performed at 20-25° C.

(c) Concentration/Diafiltration: The Capto Q ImpRes Eluate wasconcentrated to a specified concentration, then diafiltered with 10volumes of phosphate buffered saline per volume of concentrate. Theconcentration and diafiltration were performed using a 30 kDultrafiltration membrane. Once the diafiltration was completed and theconcentrate was at the desired concentration, the purified haptoglobinsolution was sterile filtered and stored at less than or equal to −20°C.

Optionally, a lipid adsorption step can be conducted before or after theanion exchange chromatography step or after theconcentration/diafiltration step. An example of a suitable lipidadsorption agent is a fumed silica like Aerosil (e.g. Aerosil 380).

The final yield of haptoglobin recovered from the process was estimatedto be approximately 0.285 g/L plasma input.

The concentrated haptoglobin preparation (approximately 2.6% w/v) wascharacterised by immune-nephelometry. Plasma proteins such as IgG, IgM,alpha-1-acid glycoprotein, pre-albumin, ceruloplasmin, hemopexin,apolipoprotein A-I, apolipoprotein B and antithrombin III were belowdetectable levels, whilst only trace amounts were detected for otherplasma proteins such as transferrin (0.099 mg/mL), alpha-1-antitrypsin(0.062 mg/mL), alpha-2-macroglobulin (0.086 mg/mL), IgA (0.65 mg/mL),and albumin (0.123 mg/mL). These results indicate that Hp accounted forat least 96% of the total protein in the preparation.

Purification of Transferrin from the Octyl 1.13M Ammonium Sulfate WashFraction:

Prior to performing ion-exchange chromatography on the Octyl WashFraction, the ammonium sulfate was diafiltered out and exchanged with alower ionic strength buffer. To this end, the Octyl Eluate wasconcentrated and diafiltered against 10 volumes of 50 mM Tris, pH 7.0per volume of eluate. The concentration and diafiltration were performedusing a 30 kD ultrafiltration membrane.

The diafiltered wash fraction was then loaded onto a GE Healthcare CaptoDEAE column equilibrated with 50 mM Tris, pH 7.0. To determine if it wasfeasible to obtain pure transferrin from this fraction, a lineargradient was performed over 10 column volumes using 50 mM Tris, pH 7.0as the starting buffer and ending with 50 mM Tris, 0.5M NaCl, pH 7.0.Fractions were collected that correspond to each peak on thechromatogram. SDS-PAGE analysis was performed to determine if one of thepeaks contained pure transferrin. As seen in FIG. 7, peak one washeavily loaded, but appeared to be pure transferrin. This indicates thatit is possible to purify transferrin from the Octyl Sepharose Washfraction, which also means that it is possible to purify hemopexin,haptoglobin, and transferrin from the same starting material (see alsoFIG. 10).

1. A method of purifying haptoglobin and hemopexin from a solutioncontaining both proteins, the method comprising: (i) providing asolution containing both haptoglobin and hemopexin; (ii) precipitatingthe haptoglobin from the solution by adding ammonium sulphate to thesolution; (iii) separating the precipitated haptoglobin from thesolution containing hemopexin; and (iv) separately purifying thehaptoglobin and/or hemopexin in one or more steps.
 2. The method ofclaim 1 wherein the haptoglobin is precipitated from the solution byadding about 2.2M to about 2.5M ammonium sulphate to the solution. 3.The method of claim 2 wherein the haptoglobin is precipitated from thesolution by adding about 2.4M ammonium sulphate to the solution.
 4. Themethod of claim 1, wherein the haptoglobin is precipitated from thesolution at a pH of less than or equal to
 8. 5. The method of claim 4,wherein the haptoglobin is precipitated from the solution at a pH withinthe range of about 6 to about
 8. 6. The method of claim 5, wherein thehaptoglobin is precipitated from the solution at a pH of about
 7. 7. Themethod of claim 1, wherein the solution containing both haptoglobin andhemopexin further comprises transferrin.
 8. The method of claim 1,wherein the solution is a human plasma fraction.
 9. The method of claim8, wherein the solution is a Cohn Fraction IV.
 10. The method of claim9, wherein the solution is a Cohn Fraction IV₄.
 11. The method of claim1, wherein the method further comprises: (i) dissolving the precipitatedhaptoglobin in a buffer to obtain a haptoglobin solution; (ii) passingthe haptoglobin solution through a strong anion exchange chromatographicresin under conditions such that the haptoglobin binds to the resin;(iii) eluting the haptoglobin from the resin; and (iv) recovering theeluted haptoglobin.
 12. The method of claim 11, further comprisingexposing the haptoglobin solution to a lipid removal agent underconditions which allow lipid to bind to the agent.
 13. The method ofclaim 1, wherein the method further comprises: (i) passing the solutioncontaining hemopexin through a hydrophobic interaction chromatographicresin under conditions that allow the hemopexin to bind to the resin;(ii) collecting the flow-through fraction from step (i); (iii)optionally washing the resin following step (ii) and collecting theflow-through wash fraction; and (iv) eluting the hemopexin from theresin following step (ii) and/or following step (iii); and (v)recovering the eluted hemopexin.
 14. The method of claim 13, furthercomprising: (i) passing the eluted hemopexin through a metal ionaffinity chromatographic resin under conditions that allow the hemopexinto bind to the resin; (ii) eluting the hemopexin from the resin; and(iii) recovering the eluted hemopexin.
 15. The method of claim 7 furthercomprising: (a) passing the solution containing hemopexin through ahydrophobic interaction chromatographic resin under conditions thatallow the hemopexin to bind to the resin; (b) collecting theflow-through fraction from step (a); (c) optionally washing the resinfollowing step (b) and collecting the flow-through wash fraction; (d)passing the flow-through fraction from step (b) and/or the flow-throughwash fraction from step (c) through a weak anion exchangechromatographic resin under conditions such that transferrin binds tothe resin; and (e) recovering the transferrin from the resin.
 16. Acomposition comprising the haptoglobin recovered by the method ofclaim
 1. 17. A composition comprising the hemopexin recovered by themethod of claim
 1. 18. A composition comprising the transferrinrecovered by the method of claim
 15. 19. A formulation comprising thecomposition of claim 16 and a pharmaceutically acceptable carrier.
 20. Amethod of treating a condition associated with haemolysis, the methodcomprising administering to a subject in need thereof the composition ofclaim
 16. 21. The method of claim 20, wherein the condition is selectedfrom haemolytic anaemia, transfusion-induced haemolysis, haemolyticuraemic syndrome, an autoimmune disease, malaria infection and burns.22. The method of claim 20, wherein the condition is selected fromsickle cell anaemia, hereditary spherocytosis, hereditaryelliptocytosis, thalassemia, congenital dyserythropoietic anemia andParoxysmal nocturnal hemoglobinuria, systemic lupus erythematosus andchronic lymphocytic leukemia. 23.-25. (canceled)
 26. A method oftreating a condition associated with haemolysis, the method comprisingadministering to a subject in need thereof the composition of claim 17.27. The method of claim 26, wherein the condition is selected fromhaemolytic anaemia, transfusion-induced haemolysis, haemolytic uraemicsyndrome, an autoimmune disease, malaria infection and burns.
 28. Themethod of claim 26, wherein the condition is selected from sickle cellanaemia, hereditary spherocytosis, hereditary elliptocytosis,thalassemia, congenital dyserythropoietic anemia and Paroxysmalnocturnal hemoglobinuria, systemic lupus erythematosus and chroniclymphocytic leukemia.
 29. A method of treating a condition associatedwith haemolysis, the method comprising administering to a subject inneed thereof the composition of claim
 18. 30. The method of claim 29,wherein the condition is selected from haemolytic anaemia,transfusion-induced haemolysis, haemolytic uraemic syndrome, anautoimmune disease, malaria infection and burns.
 31. The method of claim29, wherein the condition is selected from sickle cell anaemia,hereditary spherocytosis, hereditary elliptocytosis, thalassemia,congenital dyserythropoietic anemia and Paroxysmal nocturnalhemoglobinuria, systemic lupus erythematosus and chronic lymphocyticleukemia.